CPT-TCPP polymeric nanoplatform for sonodynamic-chemotherapy synergistic tumor therapy through ferroptosis-apoptosis dual cell death reprogramming

Abstract

Traditional cancer therapies are constrained by tumor diversity, therapeutic resistance, and side effects, thereby necessitating the development of precise multimodal antitumor strategies. This work reports a relatively high-drug-loading polymeric nanoplatform, PSCT, for combined sonodynamic therapy and chemotherapy. Camptothecin (CPT) and tetracarboxyphenylporphyrin (TCPP) were covalently linked to a polymer and self-assembled into stable nanoparticles of about 140 nm. PSCT enabled efficient co-delivery of both agents and favored tumor accumulation. In the reductive tumor microenvironment, elevated glutathione triggered disulfide cleavage and drug release. Under ultrasound (US) irradiation, TCPP generated ROS, resulting in lipid peroxidation and mitochondrial impairment. RNA-Seq analysis and molecular dynamics simulations indicated that PSCT combined with US reprogrammed a dual cell death network involving apoptosis and ferroptosis, accompanied by increased Cleaved Caspase-3 and decreased GPX4 expression. In CT26 tumor-bearing mice, PSCT showed efficient tumor accumulation and achieved an 85.19% ± 5.25% tumor inhibition rate without obvious systemic toxicity. This nanoplatform synergizes sonodynamic therapy and chemotherapy, overcoming resistance from single-pathway treatments and providing a low-toxicity strategy for the precise management of deep-seated tumors.

Graphical abstract

A glutathione-responsive polymeric nanoplatform, PSCT, was self-assembled from Poly-SS-TCPP and PEG-SS-CPT for high-loading co-delivery of sonosensitizer and camptothecin. After tumor accumulation, GSH-triggered disulfide cleavage releases CPT, while ultrasound-activated TCPP amplifies ROS generation. PSCT induces Caspase-3 activation and GPX4 downregulation to co-induce apoptosis and ferroptosis, enabling dual-pathway regulation to overcome cell death resistance and enhance antitumor efficacy.

1. Introduction

Malignant tumors have persistently high global incidence and mortality, with an annual upward trend and younger age at onset, posing a major global public health challenge [1]. Globally, cancer accounts for nearly one-sixth of deaths, a quarter of all noncommunicable disease (NCD)-related mortality, and 30% of premature NCD deaths in adults aged 30-69 years [2]. At present, surgery, radiotherapy, and chemotherapy remain the main clinical treatment options for cancer. However, these strategies still face several major limitations [3]. These include marked tumor heterogeneity, frequent multidrug resistance, prominent systemic toxic side effects, and unsatisfactory efficacy against deep-seated solid tumors [4]. Consequently, the development of efficacious, low-toxicity, and precision-targeted antitumor strategies has become an urgent clinical imperative.

Among emerging anticancer approaches, reactive oxygen species (ROS)-based interventions have attracted considerable attention as a way to overcome the shortcomings of conventional therapies [5]. Substantial evidence has revealed the crucial molecular functions of ROS in tumor progression [6,7]. As key regulators of redox homeostasis and cell death signaling, ROS can activate multiple death pathways, including apoptosis and ferroptosis, thereby influencing the tumor microenvironment (TME) [8,9]. However, the clinical translation of ROS-based monotherapy remains challenging. On the one hand, the highly upregulated antioxidant defense system in tumor cells rapidly scavenges intracellular ROS, significantly compromising therapeutic efficacy [10,11]. On the other hand, therapies that depend on a single death pathway are prone to resistance, making complete tumor eradication highly difficult [12,13]. These limitations suggest that the concurrent induction of apoptosis and ferroptosis may offer a more effective antitumor strategy.

In recent years, several ROS-generating therapeutic modalities have developed rapidly, including chemodynamic therapy (CDT) [14], photodynamic therapy (PDT) [15], and sonodynamic therapy (SDT) [16,17]. However, each modality has inherent limitations. Previous studies have demonstrated that CDT is limited by insufficient endogenous hydrogen peroxide (H₂O₂) within tumors and low catalytic efficiency, which prevents it from generating ROS [18,19]. PDT is limited by the shallow tissue penetration depth of light and tumor microenvironment hypoxia [20]. By comparison, SDT has distinct advantages, including deep tissue penetration, noninvasiveness, and favorable biosafety, which make it highly suitable for treating deep-located solid tumors [21,22]. Chen et al. reported that SDT combined with temozolomide (TMZ) inhibited the migration of C6 glioma cells and promoted mitochondria-mediated apoptosis by suppressing the expression of sodium-hydrogen exchanger 1 (NHE-1) [23]. Therefore, a rationally designed combination of SDT and chemotherapy may produce synergistic antitumor effects through complementary mechanisms, effectively counteract tumor resistance, and improve treatment results.

Camptothecin (CPT), a classic chemotherapeutic agent, triggers tumor cell apoptosis by inducing DNA double-strand breaks [24]. It has been widely applied in the clinical management of various malignancies, including triple-negative breast, liver, and colorectal cancers [25,26]. However, translating ROS-based monotherapy into clinical applications remains severely constrained by some limitations, including poor aqueous solubility, insufficient in vivo stability, and pronounced off-target toxicity [27]. Tetracarboxyphenylporphyrin (TCPP) is a classic sonosensitizer with excellent ROS generation efficiency [28], yet its clinical potential is restricted by its lack of tumor-targeting ability and limited efficacy as a monotherapy [29]. Nanodelivery systems provide an effective way to address these drawbacks. Through the Enhanced Permeability and Retention (EPR) effect, they enable targeted tumor accumulation and realize stimuli-responsive, site-specific drug release [30]. Furthermore, increased glutathione (GSH) concentrations in tumor tissues can act as a specific trigger signal [31]. Through the disulfide bond structure, nanoparticles maintain high stability during systemic circulation, while undergoing rapid disassembly and drug release within tumor tissues [32]. Moreover, the combination of SDT-generated ROS and GSH depletion can further disrupt redox homeostasis, potently activate the ferroptosis pathway, and provide a potential strategy to complement apoptosis-based tumor cell killing [33].

Notably, high drug loading capacity (DLC) is a core determinant of the clinical efficacy and biosafety of nanomedicines. Compared with some physically encapsulated TCPP-based nanoplatforms, covalent drug incorporation can increase the proportion of active therapeutic components and reduce premature leakage during systemic circulation [34]. High DLC can significantly increase the amount of active drug delivered to tumors, helping the local concentration reach an effective therapeutic level. At the same time, it reduces the proportion of inert carrier materials, thereby lowering potential carrier-related toxicity and improving in vivo delivery efficiency and biocompatibility [35]. Conversely, low drug loading leads to insufficient tumor drug exposure, poor efficacy, extra safety risks from excess carriers, and restricted clinical translation [36]. Thus, constructing a high-loading, microenvironment-responsive cooperative delivery system is a critical prerequisite for efficient and low-toxic tumor therapy.

In the present work, we established a redox-responsive multifunctional nanosystem with relatively high active-agent content, termed PSCT. The sonosensitizer TCPP and the chemotherapeutic agent CPT were covalently linked to polymer backbones to form Poly-SS-TCPP and PEG-SS-CPT, respectively, followed by self-assembly into nanoparticles (Scheme 1). PSCT was designed to accumulate in tumors through the EPR effect and release its payload in response to the GSH-rich tumor microenvironment. Under ultrasound (US) irradiation, TCPP generated ROS to amplify oxidative stress and promote ferroptosis, whereas CPT induced apoptosis through DNA damage-associated signaling, accompanied by Caspase-3 activation. Consequently, these features endowed PSCT with synergistic sonodynamic-chemotherapeutic activity. The system significantly enhanced anti-tumor efficacy while minimizing toxic side effects, and may offer a promising approach for the precise and low-toxicity therapy for deep-seated solid tumors.

Scheme 1.

Structural composition and anti-cancer mechanism of PSCT. Self-assembled from Poly-SS-TCPP and PEG-SS-CPT, PSCT accumulates at tumor sites. Upon GSH-triggered disulfide cleavage and US irradiation, it synergistically induces Apoptosis and Ferroptosis via Caspase-3 activation and GPX4 inhibition, achieving enhanced efficacy with minimal toxicity.

2. Methods

2.1. Reagents and materials

GSH, CPT, TCPP, poly (ethylene glycol) monomethyl ether (molecular weight 5000), poly (ethylene glycol) (molecular weight 300), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), Tween 20, Tween 80, Fer-1, Z-VAD-FMK, coumarin 6 (C6), and DIR [DiIC18(7)] were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Cleaved Caspase-3 polyclonal antibody and GPX4 monoclonal antibody were purchased from Wuhan Proteintech Group. Easy PAGE gel preparation kit, TUNEL apoptosis detection kit, and ready-to-use DAPI staining solution (10 μg/mL) were procured from Sevenbio (Beijing) Biotechnology Co., Ltd. Glass-bottom culture dishes were purchased from Jet Biofil. CCK-8 kit was acquired from Dalian Meilun Biotechnology Co., Ltd. Calcein-AM/PI cell kit, ROS detection kit, ATP assay kit, and SDS-PAGE protein loading buffer (5×) were sourced from Shanghai Beyotime Biotechnology Co., Ltd. CT26 cells were obtained from Wuhan Pricella Life Science Co., Ltd. RPMI-1640 medium was purchased from Sunncell Biotechnology (Wuhan, China). Male Balb/c mice (6-8 weeks of age, 20-22 g) were purchased from Changsheng Biotechnology (Liaoning). All animal experiments in the present study were conducted in strict compliance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of Dalian Medical University (approval number: XL2603170665).

2.2. Preparation of nanoparticles

Poly-SS-TCPP and PEG-SS-CPT were mixed at a mass ratio of 1:3 and dissolved in an appropriate volume of chloroform. The mixture was then subjected to rotary evaporation to yield a uniform thin film, followed by vacuum drying for 12 h. After drying, PBS was added to rehydrate the film, yielding PSCT nanoparticles. PSCT@C6 and PSCT@DIR nanoparticles were prepared following an identical procedure, with the respective fluorescent probes incorporated during the film formation step.

2.3. Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). A p < 0.05 was considered statistically significant (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). Additional methodological details are available in the Supplementary Materials.

3. Results and discussion

3.1. Preparation and characterization of nanoplatform

To construct tumor microenvironment (TME)-responsive multifunctional nanoparticles (PSCT), two amphiphilic molecules, Poly-SS-TCPP and PEG-SS-CPT, were synthesized in this study (Figs. S1–6). The drug contents of the two polymer conjugates were determined by UV-visible (UV-Vis) spectrophotometry. The TCPP content in Poly-SS-TCPP and the CPT content in PEG-SS-CPT were calculated to be 35.8% and 12.0%, respectively. PSCT nanoparticles were subsequently formed through the hydrophilic-hydrophobic self-assembly of Poly-SS-TCPP and PEG-SS-CPT at a mass ratio of 1:3. Unlike conventional physically encapsulated nanosystem, PSCT integrates the therapeutic agents into the nanoparticle framework through covalent conjugation rather than loading them as cargo. This design can reduce premature leakage or phase separation between the drug and carrier.

To contextualize the loading performance, we compared our system with previously reported GSH/redox-responsive or TCPP-containing chemo-sonodynamic nanoplatforms. The TCPP loading contents in representative systems were generally below 30% [37,38]. In comparison, the TCPP content in Poly-SS-TCPP in this work reached 35.8%, indicating efficient covalent incorporation of TCPP into the polymer framework. These results suggest that our covalent drug-integrated design provides a relatively high active-agent content while reducing the proportion of inert carrier materials.

Transmission electron microscopy (TEM) graph showed that PSCT displayed a uniform spherical morphology with a narrow size distribution and no obvious aggregation, indicating the successful formation of well-defined nanoparticles (Fig. 1A). As shown in Fig. 1B, PSCT markedly improved the aqueous dispersibility of both TCPP and CPT. Dynamic light scattering (DLS) analysis showed that PSCT had a Zeta potential of −28.6 ± 0.8 mV (Fig. S7), an average hydrodynamic diameter of 140.0 ± 0.8 nm, and a polydispersity index (PDI) of 0.31 ± 0.04 (Fig. 1C), indicating good colloidal uniformity. UV-Vis spectra confirmed that PSCT retained the characteristic absorbance peaks of both CPT and TCPP, confirming their successful incorporation into the nanosystem (Fig. 1D). To evaluate the requirements of systemic in vivo administration, this study monitored the particle size and PDI of PSCT under storage and physiological conditions to evaluate their stability. The particle size and PDI of PSCT remained stable during 4 days of storage at 4°C, but increased on day 5 (Fig. 1E and F). Furthermore, these two parameters remained stable during incubation in medium containing 10% FBS for up to 48 h (Fig. 1G and H). The results indicate that PSCT possess good stability and can meet the requirements for intravenous administration in vivo.

Fig. 1.

(A) TEM image of PSCT composite nanoparticles. (B) Photographs of PSCT, CPT, and TCPP solutions. (C) Hydrodynamic size distribution of PSCT measured by DLS (n = 3). (D) UV-Vis absorption spectra of PSCT. (E) Changes in the hydrodynamic size of PSCT over 5 days. (F) Changes in the PDI of PSCT over 5 days (n = 3). (G) Changes in the hydrodynamic size of PSCT in 10% FBS within 48 h (n = 3). (H) Changes in the PDI of PSCT in 10% FBS within 48 h (n = 3). (I) Hydrodynamic size distribution of PSCT in 10% FBS (n = 3). (J) TEM image of PSCT under high GSH conditions. (K) In vitro cumulative release profile of CPT from PSCT (n = 3). (L) Detection of singlet oxygen generation ability and oxidative stress levels under US stimulation. ***p < 0.001.

Effective nanotherapeutics need to release drug within the TME to reduce systemic toxicity and enhance antitumor efficacy. In tumor cells, high intracellular GSH levels can cleave the disulfide bonds in PSCT, causing the nanoparticles to disassemble and release native CPT, which promotes tumor cell apoptosis and increases intracellular oxidative stress. Accordingly, the changes in particle size, PDI, morphology, and drug release characteristics of PSCT were evaluated. After exposure to high GSH levels, the structural integrity of PSCT was disrupted, resulting in an increased particle size of 209.1 ± 8.3 nm and a PDI of 0.62 ± 0.03 (Fig. 1I and J), indicating GSH-responsive disassembly. In vitro drug release experiments demonstrated that CPT was released from PSCT in a GSH-responsive manner. At pH 5.5 with 5 mM GSH, CPT release reached nearly 70% and plateaued around 80 h (Fig. 1K). In contrast, release under physiological conditions remained low. Under physiological conditions (pH 7.4), the cumulative TCPP release is only about 4%, indicating negligible TCPP leakage from PSCT in the normal physiological environment (Fig. S8). These results suggest that PSCT can selectively release the chemotherapeutic drug and sonosensitizer in the tumor microenvironment, supporting US-activated chemo-sonodynamic therapy.

Since PSCT contains the sonosensitizer TCPP, which can generate singlet oxygen upon US stimulation, the SOSG probe was employed to investigate whether PSCT could produce singlet oxygen under US irradiation. The results revealed that PSCT treatment caused a clear increase in SOSG fluorescence intensity upon US irradiation (Fig. 1L), indicating that PSCT could efficiently generate singlet oxygen and raise oxidative stress levels in response to US stimulation. These findings were further corroborated by the results of electron spin resonance (ESR) analysis (Fig. S9).

3.2. Antitumor activity of PSCT + US in vitro

To investigate the antitumor activity of PSCT in vitro, the cellular uptake of PSCT by CT26 cells was examined. CT26 cells were treated with C6-loaded PSCT nanoparticles (PSCT@C6) and observed. As shown in Fig. 2A and Fig S10-11, the intracellular green fluorescence gradually increased with prolonged incubation (0, 1, 2, and 4 h), and the magnification confocal images further provide clearer evidence for intracellular uptake, suggesting that PSCT can be efficiently internalized by CT26 cells and accumulate intracellularly. Quantitative analysis of integrated optical density (IOD) further confirmed a time-dependent increase in intracellular C6 accumulation (Fig. 2B). These findings confirmed that PSCT were efficiently taken up by colorectal cancer cells, providing a basis for subsequent intracellular drug release and multimodal therapeutic action.

Fig. 2.

(A) Fluorescence microscopy images showing the cellular uptake of nanoparticles by CT26 cells at different time points (n = 3). (B) Quantitative analysis of intracellular C6 fluorescence intensity at different time points (n = 3). (C) Viability of CT26 cells after 24 h incubation with different formulations (n = 6). (D) Live/dead cell staining of CT26 cells after 24 h treatment with different formulations (n = 3). (E) Fluorescence inverted microscopy images of intracellular ROS generation in CT26 cells treated with different formulations (n = 3). (F) Quantitative fluorescence analysis of Fig. 2E. (G) Fluorescence imaging of the redox status in different treatment groups (n = 3). (H) Quantitative analysis of LPO concentration in each group. (I) TEM images of CT26 cells after 24 h incubation with Control and PSCT + US groups. *p < 0.05; **p < 0.01; ***p < 0.001.

The antiproliferative effect of PSCT was then evaluated using the CCK-8 assay. As shown in Fig. 2C, PSCT showed delayed cytotoxicity, likely due to GSH-triggered disulfide bond cleavage and drug release. Upon US irradiation, PSCT exhibited markedly enhanced cytotoxicity, with the lowest IC50 value of 1.1 μg/mL. Additional CCK-8 assays using CPT, TCPP + US, and CPT + TCPP + US at equivalent doses showed IC50 values of 5.9, 14.4, and 3.2 μg/mL, respectively (Fig. S12). Based on the cell viability data, the interaction was further quantified using the Chou-Talalay method by calculating the combination index (CI). The calculated CI value is 0.89 (below 1), indicating a synergistic interaction between chemotherapy and sonodynamic therapy. Moreover, PSCT + US showed stronger cytotoxicity than CPT + TCPP + US, probably due to improved cellular uptake, co-delivery of CPT/TCPP, and GSH-responsive release. The trend was further validated by the live/dead cell double staining assay. As shown in Fig. 2D, the PSCT + US group showed an almost entirely PI-positive dead cell population within the field of view, demonstrating the potent killing capacity of the multimodal combination therapy.

To investigate the mechanism underlying the enhanced cytotoxicity, the DCFH-DA fluorescent probe was utilized to monitor the level of intracellular ROS generation. As shown in Fig. 2E–F and Fig. S13, only weak fluorescence was detected in the Control groups. However, a marked elevation in intracellular ROS level was observed in the PSCT-treated group, which was further amplified after US exposure. These results indicate that US activation of TCPP amplified oxidative stress in tumor cells. Because excessive ROS can trigger ferroptosis through lipid peroxide accumulation, LPO levels were further examined. LPO staining and quantitative analysis showed a pronounced increase in lipid peroxidation in the PSCT + US group, supporting the induction of ferroptosis (Fig. 2G and H). The observed trend matched the ROS results completely, indicating that PSCT + US effectively promoted oxidative damage at the lipid level. Under the present ultrasound conditions, US alone caused no obvious changes in CT26 live/dead staining, intracellular ROS, or LPO levels (Figs. S14–16), indicating that the cytotoxicity of PSCT + US was mainly mediated by TCPP-based sonodynamic action. Future studies will incorporate flow cytometry to quantitatively assess cell death, ROS generation, and lipid peroxidation.

Mitochondria are major targets of lipid peroxidation damage during ferroptosis. Therefore, mitochondrial ultrastructure was observed via Bio-TEM. As shown in Fig. 2I, cells in the control group showed mitochondria with a typical elongated shape, maintaining a complete bilayer membrane and well-organized internal cristae. In contrast, mitochondria showed marked swelling and vacuolization, with fractured or even completely disappeared internal cristae in the PSCT + US treatment group. The distinctive pathological changes in mitochondria offered morphological proof of a direct attack on the mitochondrial membrane due to significant LPO buildup. Taken together, the above results show that PSCT + US promotes lipid peroxidation, damages mitochondria, and enhances tumor cell killing through the combined induction of ferroptosis and apoptosis.

3.3. Tumor targeting and biosafety evaluation of PSCT + US in vivo

Efficient tumor accumulation is essential for PSCT to exert its antitumor activity in vivo. To evaluate its biodistribution, PSCT@DIR nanoparticles were prepared and intravenously injected into tumor-bearing mice. In vivo biodistribution behavior was dynamically monitored using a small animal imaging system. As shown in Fig. 3A, the tumor fluorescence signal of PSCT@DIR gradually increased over time and reached the highest level at 24 h, indicating effective tumor accumulation and retention. Notably, clear tumor accumulation was observed at 6 h, with fluorescence intensity comparable to that at 8 h, suggesting sufficient accumulation for ultrasound treatment. In contrast, free DIR showed broad whole-body distribution without obvious tumor enrichment. Quantitative analysis further confirmed the stronger and time-dependent tumor accumulation of PSCT@DIR compared with free DIR (Fig. 3B). Therefore, 6 h post-injection was selected as the ultrasound irradiation time point for subsequent therapy. Future studies will further optimize the irradiation timing through extended imaging and therapeutic comparisons at different time windows.

Fig. 3.

(A) In vivo fluorescence imaging of tumor-bearing mice after tail vein injection of DIR and PSCT@DIR (n = 5). (B) Quantitative fluorescence analysis of Fig. 3A. (C) Ex vivo fluorescence imaging of major organs and tumors (n = 5). (D) Quantitative fluorescence analysis of Fig. 3C (n = 5). (E) Schematic illustration of the acute toxicity experiment in mice. (F) Hemolysis rates of PEG-SS-CPT and Poly-SS-TCPP at different concentrations (n = 3). (G) Photographs of hemolysis assays. (H) H&E staining of major organs after tail vein injection of different concentrations of PSCT composite nanoparticles for 24 h (n = 3). (I) Blood biochemical parameters of mice after tail vein injection of different concentrations of PSCT composite nanoparticles for 24 h (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001.

At 24 h after injection, mice were euthanized to collect the main organs and tumor tissues. As shown in Fig. 3C and D, the tumor fluorescence signal in the PSCT@DIR markedly exceeded that of the DIR group. Quantitative data further confirmed that PSCT exhibited prominent tumor accumulation, with far higher fluorescence intensity in tumor tissues than that in all normal organs including the heart, liver, spleen, lung and kidney. The above results confirm that PSCT can passively accumulate in tumors through the EPR effect, thereby reducing nonspecific distribution in normal tissues.

After confirming tumor-targeting ability in vivo, the hemocompatibility evaluation and optimal in vivo antitumor dose determination were performed. Mouse red blood cells were incubated with PEG-SS-CPT or Poly-SS-TCPP at 50 to 400 μg/mL. As shown in Fig. 3E–G, both materials exhibited hemolysis rates below 5% at all tested concentrations, comparable to the saline control, indicating good hemocompatibility for intravenous administration. Then, BALB/c mice were assigned to four groups and injected with normal saline or PSCT at doses of 3, 6, and 9 mg/kg (calculated according to CPT and TCPP equivalent doses). H&E staining of major organs revealed no obvious histopathological abnormalities or tissue damage in any PSCT-treated group (Fig. 3H). Serum biochemical analysis further confirmed that ALT, AST, Cr, and BUN levels in the 6 mg/kg group were not significantly different from those in the control, whereas elevated Cr values were observed in 9 mg/kg, consistent with the pathological findings (Fig. 3I). Based on the above results, the equivalent dose of 6 mg/kg for CPT and TCPP was chosen as the administration dose for subsequent in vivo antitumor experiments.

After confirming its in vivo biosafety, the pharmacokinetic behavior of PSCT was further investigated. Pharmacokinetic studies were performed using free drugs and PSCT at a dose of 6 mg/kg. As shown in Tables 2–3 in the Supplementary material, PSCT markedly prolonged systemic blood circulation relative to the free CPT + TCPP combination. For CPT, PSCT delivery yielded a substantially higher peak plasma concentration (Cmax) of 4.79 mg/L, approximately 11-fold higher than the 0.44 mg/L detected for free CPT. The 24-h area under the plasma concentration-time curve (AUC₀-_24h) was also markedly increased, from 2.52 to 58.61 mg h/L. In addition, PSCT extended the terminal elimination half-life (t₁/₂) of CPT from 8.37 to 11.82 h, reduced its apparent clearance (CL) from 2.24 to 0.08 L/h/kg, and increased the mean residence time (MRT_0-24h), indicating slower blood clearance. Similarly, for TCPP, PSCT likewise increased C_max, AUC_0–24h, and MRT_0–24h, while reducing apparent CL. The shorter apparent t_1/2 of TCPP in the PSCT group was likely due to the limited 24 h sampling window, which may not fully reflect the true terminal elimination phase of the long-circulating nanosystem. Overall, these results indicate prolonged circulation and enhanced systemic exposure of PSCT, consistent with its gradual tumor accumulation observed in vivo.

3.4. Antitumor efficacy and biosafety of PSCT + US in vivo

Based on the evaluations, the antitumor efficacy of PSCT + US was systematically assessed in a CT26 tumor-bearing mouse model. As shown in Fig. 4A, a subcutaneous CT26 tumor-bearing model was established by axillary injection of CT26 cells into BALB/c mice. When tumor volumes reached approximately 85 mm³, the mice were assigned to four groups (n = 5): (1) Control; (2) CPT + TCPP; (3) PSCT; and (4) PSCT + US. For the PSCT + US group, US irradiation (1 MHz, 1.5 W/cm², 5 min) was applied at 6 h after each administration. To exclude the potential contribution of ultrasound-induced thermal effects, the local tumor temperature was monitored during in vivo US irradiation. The temperature increased only a slight increase from 32.0°C to 33.3°C, which remained below the threshold for obvious thermal damage (Fig. S17). These results suggest that the antitumor effect was mainly attributed to TCPP-mediated ROS generation rather than hyperthermia. As shown in Fig. 4B and C, the tumors in the control showed continuous and rapid growth during the treatment period, with a significantly larger average volume at the end of treatment than in all treatment groups. The CPT + TCPP group exhibited a moderate tumor inhibitory effect, with a slower tumor growth rate compared to untreated controls. The PSCT showed a further enhanced antitumor effect, which was superior to the free drug group, attributable to the passive tumor targeting accumulation and intracellular GSH-responsive drug release mechanism of the nanoparticles. Tumor growth was most effectively suppressed in the PSCT + US group, with extremely slow tumor growth throughout the treatment course.

Fig. 4.

(A) Construction of CT26 tumor-bearing mice and schematic diagram of drug administration. (B-C) Change curve of tumor volume of mice in each treatment group during treatment (n = 5). (D) Relative tumor weight of mice in each treatment group after treatment (n = 5). (E) Tumor inhibition rate of each treatment group. (F) Body Weight of each treatment group (n = 5). (G) HE staining of organs. (H) Blood biochemical analysis indicators of mice in each group after treatment (n = 5). (I) HE staining of the tumor after treatment. (J) TUNEL staining of the tumor after treatment. (K) Fluorescence quantitative images of TUNEL assay (n = 3). PBS as a control. *p < 0.05; **p < 0.01; ***p < 0.001.

After the treatment, the PSCT + US group exhibited a significantly lower tumor weight than all other treatment groups, with a sharp reduction in relative tumor weight (Fig. 4D). Tumor inhibition rate quantification revealed that PSCT + US treatment yielded an inhibition rate of 85.19% ± 5.25%, significantly superior to those of the PSCT group and the free drug group (Fig. 4E). This value is comparable to or higher than those reported for several chemo/SDT combination platforms, suggesting the favorable antitumor efficacy of PSCT + US [39]. Collectively, these results indicate that US irradiation enhanced the antitumor effect of PSCT, likely through chemo-sonodynamic synergy, which will be further validated using more comprehensive in vivo control groups in future studies.

The safety of the treatment in vivo was also examined. Body weight was recorded throughout the treatment period, and no significant body weight loss was detected (Fig. 4F). The results showed that the tested treatments did not cause obvious systemic toxicity and suggesting good biocompatibility in vivo. In addition, the major organs were collected for H&E staining and serum biochemical analysis. As shown in Fig. 4G and H, histological examination revealed no significant tissue damage or pathological abnormalities in the main organs of mice in the PSCT + US group. Serum levels of ALT, AST, Cr, and BUN did not differ significantly among the groups, suggesting that none of the treatments induced apparent hepatic or renal toxicity. Notably, compared with several reported CPT-based nanoplatforms, PSCT achieved effective antitumor efficacy at a relatively moderate administration dose while maintaining favorable biosafety [40,41]. To further assess the safety of repeated PSCT administration, a 14-day toxicity study was conducted at the therapeutic dose. As shown in Fig. S18A–C, mice showed stable body weights, no obvious organ damage in H&E staining, and no significant abnormalities in serum biochemical indicators. Nevertheless, additional evaluation under higher-dose repeated-administration conditions is still warranted and will be included in future studies. These results demonstrated that the combination therapeutic strategy maintained favorable in vivo biosafety while exerting potent antitumor efficacy.

Histopathological examination of tumor tissues provided further evidence of therapeutic efficacy (Fig. 4I–K). Tumor tissues from the free drug group and the PSCT group showed varying degrees of structural loosening and nuclear pyknosis, with enlarged TUNEL-positive areas. In the PSCT + US group, the tumor tissue architecture was further disrupted, and TUNEL-positive signals were further expanded, indicating that the US-synergized treatment induced the most pronounced tumor cell apoptosis. In summary, PSCT + US produced marked antitumor effects in CT26 tumor-bearing mice while maintaining satisfactory in vivo safety.

3.5. Antitumor mechanism of PSCT + US

To investigate the effect of PSCT + US treatment on the cellular transcriptome, RNA sequencing analysis was performed on cells from the control and PSCT + US treatment groups. As shown in Fig. 5A, principal component analysis (PCA) clearly separated the two groups along PC1 (52.69%) and PC2 (17.41%), while biological replicates within each group clustered closely together. This result indicates good reproducibility and substantial transcriptomic changes induced by PSCT + US treatment. Violin plots and Pearson correlation analysis also showed no obvious outliers, ruling out interference from sample heterogeneity and batch effects (Fig. S19A–B). Based on the screening threshold (|log₂FC| ≥ 1, FDR < 0.05), a total of 4107 differentially expressed genes (DEGs) were screened out, including 2141 upregulated genes and 2096 downregulated genes (Fig. 5B). The DEG heatmap further showed a clear difference in gene expression patterns between the two groups, confirming that PSCT + US induced extensive transcriptomic remodeling (Fig. 5C). Gene Ontology (GO) functional enrichment analysis (Fig. 5D, Fig. S19C) revealed significant modulation of processes related to redox regulation, ionic balance, and apoptotic cascades, implying the initiation of cell death by disrupting cellular antioxidant defense. KEGG pathway enrichment (Fig. 5E, Fig. S19D) showed marked enrichment of cell death pathways, particularly ferroptosis and apoptosis. Subsequent mechanistic investigation was therefore focused on these two programmed cell death modalities. Focusing on the core scientific question of this study, subsequent mechanistic dissection was concentrated on the two programmed cell death pathways, ferroptosis and apoptosis. Gene Set Enrichment Analysis (GSEA) (Fig. 5F) revealed significant enrichment of upstream signaling pathways including MAPK, PI3K-Akt, p53 and Ras, laying a molecular foundation for the activation of downstream cell death signaling. Further GSEA validation of the ferroptosis and apoptosis pathways (Fig. 5G and H) showed prominent activation of ferroptosis, accompanied by disrupted iron homeostasis and suppressed glutathione metabolism. Meanwhile, intrinsic, extrinsic and endoplasmic reticulum stress-related apoptotic pathways were simultaneously upregulated, supporting the synergistic initiation of dual cell death programs by PSCT + US. The key gene expression heatmap (Fig. 5I) further verified these alterations. In the ferroptosis pathway, anti-ferroptosis genes including Gpx4, Slc39a8, Slc3a2 and Slc7a11 were markedly downregulated, whereas pro-ferroptosis Acsl4 and Trp53 were upregulated. In the apoptosis pathway, pro-apoptotic genes (Casp1, Bax, Pidd1, Casp12, Bcl2l15) were increased, while anti-apoptotic Bcl2 and Bcl2l1 were decreased, consistent with GSEA results. Collectively, the above findings indicated that PSCT + US treatment remodels the cellular transcriptome and activates multiple upstream signaling networks, disrupts cellular oxidative metabolism and ion homeostasis, and coordinately induces ferroptosis and apoptosis, thereby exerting a marked inhibitory effect on cell proliferation. These suggested that the cell death induced by PSCT + US was not driven by a single target or pathway but rather represented an integrated outcome of multidimensional regulation. The involvement of multiple mechanisms may help reduce the therapeutic limitations associated with reliance on a single cell death pathway. Dual activation of ferroptosis and apoptosis may provide complementary lethal signaling. Even if tumor cells evade the killing effect of one pathway through adaptive mechanisms, the alternative pathway still exerts potent cytotoxicity, offering a new insight for overcoming tumor drug resistance.

Fig. 5.

(A) Principal component analysis (PCA) of RNA-seq data showing distinct clustering between Control and PSCT + US groups. (B) Volcano plot of differentially expressed genes (DEGs) (|Log₂FC| > 1, P < 0.05). (C) Heatmap of DEGs. (D) Bubble plot of Gene Ontology (GO) enrichment analysis for DEGs. (E) Bubble plot of KEGG pathway enrichment analysis. (F) Ridge plot displaying the enrichment landscape of key signaling pathways. (G-H) GSEA plots for (G) Ferroptosis and (H) Apoptosis. (I) Heatmap showing relative expression levels of Ferroptosis- and Apoptosis-related genes.

To explore potential protein associations that may be related to the pro-death effects of the system, target prediction analysis was conducted for camptothecin, using the SwissTargetPrediction database. Among the predicted top 20 potential targets, multiple Caspase family members (Caspase-1/3/6/7/8) were identified (Fig. 6A), suggesting that CPT may be associated with apoptosis-related signaling. Molecular docking results (Fig. 6B–D, Fig. S20A–B) showed that the binding energy of CPT with Caspase-3 reached −7.6 kcal/mol, which was notably higher than that with other family members. The docking model suggested potential hydrogen bond interactions with residues such as ARG-20 within the binding pocket, providing a possible structural basis for CPT association with Caspase-3. To further evaluate the plausibility of the docking pose, a 100-ns molecular dynamics simulation was performed. As shown in Fig. 6E–G, Fig. S20C–E, the protein backbone fluctuation (RMSF), ligand conformation (Ligand RMSD), and overall complex conformation (Complex RMSD) remained relatively stable throughout the simulation period. In addition, the radius of gyration and solvent-accessible surface area showed no major fluctuations. Free energy landscape analysis and hydrogen-bond evaluation also supported the thermodynamic stability of the CPT-Caspase-3 complex (Fig. 6H, Fig. S20F). These findings collectively suggest a possible structural basis for the association between CPT and Caspase-3 from computational perspectives. These computational observations complement the classical understanding of CPT as a topoisomerase I inhibitor. However, the docking and molecular dynamics analyses offer only computational support for a possible association, which requires further biochemical validation.

Fig. 6.

(A) The top 20 predicted target proteins by binding score. (B-D) 3D docking models of CPT bound to Caspase-7 (B) (−7.2 kcal/mol), Caspase-3 (C) (−7.6 kcal/mol), and Caspase-6 (D) (−7.3 kcal/mol). (E) RMSF of the protein backbone during 100 ns MD simulation. (F) RMSD of the ligand CPT. (G) Free energy landscape of the protein-ligand complex. (H) RMSD of the protein-ligand complex.

Based on the transcriptomic and target prediction results, key molecular changes were further validated by protein-level analysis. Western blot analysis (Fig. 7A) revealed that Cleaved Caspase-3 expression was upregulated in the PSCT + US group. This indicated that US combined with PSCT effectively activated the Caspase-3-dependent apoptotic pathway. Moreover, the ferroptosis-inhibitory protein GPX4 was substantially downregulated in the PSCT + US group compared with the control, CPT + TCPP, and PSCT groups in vitro (Fig. 7B and C). Treatment with the ferroptosis inhibitor Fer-1 obviously restored GPX4 expression, confirming that US-assisted therapy suppressed GPX4 to trigger tumor cell ferroptosis. To further evaluate ferroptosis-associated changes, intracellular GSH and Fe²⁺ levels were measured. Direct measurement after PSCT treatment was not feasible because the absorption of Poly-SS-TCPP in PSCT overlaps with the detection wavelength of the GSH assay kit, leading to spectral interference. Therefore, we used the precursor polymer Poly-SS-COOH for the assay. The results revealed a marked reduction in intracellular reduced GSH, indirectly supporting PSCT reduction response degradation and drug release in the intracellular reduction environment (Fig. S21). This strategy avoids optical interference and is also mechanistically relevant for reflecting the relationship between the disulfide structure in the PSCT system and intracellular reducing consumption. In addition, intracellular Fe²⁺ was markedly increased in the PSCT + US group (Fig. S22). Together with GPX4 downregulation, elevated LPO, and mitochondrial morphological damage, these findings support the involvement of ferroptosis-associated processes in the antitumor effect of PSCT + US. Consistently, TUNEL staining further revealed increased apoptosis-associated DNA fragmentation after PSCT + US treatment (Fig. S23). Moreover, both Fer-1 and Z-VAD-FMK partially rescued PSCT + US-induced cytotoxicity to a similar extent, suggesting that ferroptosis- and apoptosis-associated pathways jointly contributed to tumor cell killing (Fig. S24).

Fig. 7.

(A) The protein expression of Cleaved-Caspase-3 and Caspase-3 in Control, CPT + TCPP, PSCT and PSCT + US (n = 3). (B) The protein expression of GPX4 and β-actin in Control, CPT + TCPP, PSCT and PSCT + US (n = 3). (C) The protein expression of GPX4 and β-actin in Control, PSCT + US and PSCT + US + Fer-1 (n = 3). (D) Fluorescence quantitative images of Fig. 7F.(E) Fluorescence quantitative images of Fig. 7G. (F) Immunofluorescence staining of Cleaved Caspase-3 in tumors of each treatment group after treatment (n = 3). (G) Immunofluorescence staining of GPX4 in tumors of each treatment group (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Immunofluorescence staining and quantitative analysis further supported these molecular alterations. The fluorescence intensity and positive area of Cleaved Caspase-3 were substantially increased in the PSCT + US group, while GPX4 fluorescence signals were notably reduced relative to other groups (Fig. 7D–G). Consistent with transcriptomic profiling and molecular simulation data, these results demonstrated that PSCT + US exerted antitumor activity through the coordinated activation of ferroptosis and apoptotic pathways.

4. Conclusion

In this study, we developed a GSH-responsive polymeric nanoplatform, PSCT, for combined sonodynamic therapy and chemotherapy. PSCT covalently integrates CPT and TCPP, enabling high drug loading, improved dispersibility, good stability, and tumor-enriched accumulation. In the GSH-rich tumor microenvironment, PSCT undergoes disulfide bond cleavage and promotes drug release. Under ultrasound irradiation, TCPP generates ROS to induce oxidative stress, lipid peroxidation, mitochondrial damage, and ferroptosis-associated events, while CPT contributes to apoptosis-related tumor cell killing. Transcriptomic, molecular simulation, and protein analyses suggested that PSCT + US modulated ferroptosis- and apoptosis-related pathways, with GPX4 downregulation and Cleaved Caspase-3 upregulation. In CT26 tumor-bearing mice, PSCT + US achieved significant tumor inhibition with no obvious systemic toxicity. Overall, PSCT represents a redox-responsive chemo-sonodynamic nanoplatform for precise and low-toxicity treatment of deep-seated tumors.

CRediT authorship contribution statement

Lu Wang: Writing – review & editing, Writing – original draft, Visualization, Methodology. Zhibo Zhang: Methodology. Qiaoni Liu: Methodology, Investigation. Huizi Zhang: Investigation. Jinyong Peng: Writing – review & editing. Dandan Wang: Writing – review & editing.

Declaration of competing interest

The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.